Evolutionary differentiation of androgen receptor is responsible for sexual characteristic development in a teleost fish

Teleost fishes exhibit complex sexual characteristics in response to androgens, such as fin enlargement and courtship display. However, the molecular mechanisms underlying their evolutionary acquisition remain largely unknown. To address this question, we analyse medaka (Oryzias latipes) mutants deficient in teleost-specific androgen receptor ohnologs (ara and arb). We discovered that neither ar ohnolog was required for spermatogenesis, whilst they appear to be functionally redundant for the courtship display in males. However, both were required for reproductive success: ara for tooth enlargement and the reproductive behaviour eliciting female receptivity, arb for male-specific fin morphogenesis and sexual motivation. We further showed that differences between the two ar ohnologs in their transcription, cellular localisation of their encoded proteins, and their downstream genetic programmes could be responsible for the phenotypic diversity between the ara and arb mutants. These findings suggest that the ar ohnologs have diverged in two ways: first, through the loss of their roles in spermatogenesis and second, through gene duplication followed by functional differentiation that has likely resolved the pleiotropic roles derived from their ancestral gene. Thus, our results provide insights into how genome duplication impacts the massive diversification of sexual characteristics in the teleost lineage.


REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): The manuscript sounds good, designated well with enough data in the result. However, some concerns should be addressed.
1. The authors used TILLING method to knock out ara and arb, while they used CRISP/Cas9 to knock in ara and arb, which showed that they could knock out genes with CRISP/Cas9 very efficiently. In this case, why did they not knock out ara and arb with CRISP/Cas9 to check consistent with whether the mutants produced by CRISP/Cas9 had the same phenotype as the mutants produced by TILLING. We also added the following sentences to the Result section to describe that the TILLING mutations in ara and arb resulted in lack of the ligand binding domain (LBD) containing the key amino acid-substitution which generates a distinct transactivation response between Ara and Arb in vitro.
Main text, page 5, line 173-page 6, line 175, These nonsense mutations lead to expression of the truncated protein products lacking the LBD that contains a key amino acid-substitution responsible for distinct transactivation responses of Ara and Arb in vitro 30 ( Supplementary Fig. 2).
We also included the following sentences in the Supplementary Information.

Targeted gene disruption with TALENs
Genome editing mediated by transcription activator-like effector nucleases (TALENs) was performed as previously reported 2 . Ar proteins contain a N-terminal domain (NTD), DNA binding domain (DBD), and ligand binding domain (LBD) from the amino to carboxyl terminus. The TALEN pairs for ara and arb were designed to cleave the 1st exon of ara and arb at the downstream of their ATG start codons, respectively (their binding sequences are shown in Supplementary Fig. 12). Synthesis and purification of capped RNAs from the linealised TALEN expression vectors was performed using the mMessage mMachine SP6 kit (Thermo Fisher Scientific, Waltham, MA, USA) and the RNeasy Mini kit (Qiagen, Hilden, Germany). The pairs of RNA for TALENs were microinjected into fertilized medaka eggs at the 1-cell stage. F0 founders were crossed with WT fish and then germline-transmitted mutant Fish (F1) were selected by direct sequencing of the PCR products amplified from the fin clips of the adult fish using the primers, ara-F and ara-R for ara, and arb-F and arb-R for arb (Supplementary Table 1). The genetic sex of each fish was examined by genomic PCR experiments according to a previous report 3 . The mutant strains were maintained by crossing heterozygous females and males, and the resulting WT and homozygous siblings were used for phenotypic analyses. The ara and arb double heterozygous males and females (ara +/-; arb +/-) were obtained by breeding ara -/males with arb -/females. The ara and arb double heterozygous males and females were crossed to obtain males (ara -/-; arb +/-) and females (ara -/-; arb +/-). The ara and arb double homozygous males and females (ara -/-; arb -/-) were obtained by breeding males (ara -/-; arb +/-) with females (ara -/-; arb +/-). Fin and testicular phenotypes in ar TALEN KO medaka a Representative picture of the whole body and higher magnification pictures of the anal fin and dorsal fin of a WT male, ara KO male, arb KO male, and ar DKO male. The papillary processes developments were marked by red dotted circles. Arrows indicate forks in the dorsal fin.  Main text, page 10, line 335 to 337, We detected no up-regulation of ara in the arb KO and that of arb in the ara KO, which indicates that no detectable level of genetic compensation occurred between ara and arb at the whole-brain level.

Supplementary
3. In Fig.1, the pictures with only HE staining are not enough, in situ hybridization or immune-histochemistry of some gonad specific genes must be provided, such as germ cells gene vasa, Sertoli cells genes dmy、gsdf and Ledig cells gene to check why the ara and arab mutants reduce the frequency of spawning. We added the following sentences in Supplementary Information.

Section in situ hybridization
Section in situ hybridization analysis was performed as described previously 1 . The cDNAs used for preparation of antisense riboprobes of vasa, P450c17, and gsdf were cloned by standard reverse transcription polymerase chain reaction (RT-PCR) procedures using primers shown in Supplementary Table 1 We added sequences of the following primers used for cloning of gsdf, vasa, and P450c17 genes to Supplementary Table 1. gsdf-S1, TCCACCATGTCTTTGGCAC gsdf-R1, TGACCAACCCCTGCCTAC vasa-S1, ACGGCCCAAAGTGACCTAC vasa-R1, GGGTCGTAGAAGGACACGG P450c17-S1, CTCTGTGCTCCACCCTGT  Response: We appreciate the reviewer's comment. This was our mistake.
Response: We thank this careful comment. We used the British English.
8. Line 540, six month medaka are too old, they are not good for the experiment, since 3 month old medaka are matured.
Response: We appreciate the reviewer's comment.

Based on the life span analyses of medaka by long cultured data (Gopalakrishnan et
al. 2013), we judged that the 6 months old is a stably reproducing age.

Gopalakrishnan et al. (2013) reported that the plasma T and 11-KT levels peaked in
males at 8 months old and gradually decreased with age after 8 months. They also reported that the median life span of the males and females under laboratory condition was 13.7 months and 14.6 months, respectively. In consistent with these reports, in our laboratory condition, males constantly reproduce at 6 months old. In addition, as we described in the Materials and Methods, we used the male and female medaka producing fertilised eggs for at least three consecutive days until the day before the experiment were used. The ar DKO males were also reared with females in the same tank for one week until the day before the experiment.
We added the information of the age of medaka into the Materials and Methods as follows, Main text, page 17, line 596-598, We judged that the 6 months old was proper to check the testicular function of medaka, because it has been shown that the plasma T and 11-KT levels peak in male medaka at 8 months old 79 .
We added the following paper in the References. 9. Full name of 'WT' should be given as it appears for the first time.
Thank you for this comment. We added the full name before the abbreviation when it appeared for the first time in the main text line 187 (page 6), we showed WT as follows, "All wild-type (WT) and mutant fish with a Y chromosome (XY chromosomes) had testes (n = 10 in each genotype)".
Reviewer #2 (Remarks to the Author): The current manuscript analysed in detail the unique and overlapping functions of two androgen receptor (ar) ohnologs in various sexual characteristics in one of the teleost model species, the Japanese medaka. The authors used both mutants for either ar copies (ara KO and arb KO) and double mutants (ar DKO) to compare the effect resulting from the loss of either ar gene. A wide range of sexual characteristics were examined carefully, including mutant fecundity, morphology, mating success, behavour and brain gene expression patterns. With these comparisons, the authors identified distinct functions of either ar ohnolog. Furthermore, the authors used epitope-tagged AR-KI mutants show that the functional difference between the two ohnologs potentially arise from their different transcriptional regulation and intracellular localisation. Overall, this manuscript is very logically structured and easy to read with clear writing. I find the experiments conducted and analyses performed thorough and convincing. I especially enjoyed the included video of aggressive competition among the male fish. I do not have major criticism on this manuscript, but have a few comments that I hope the authors can address to further improve the manuscript. I couldn't find information regarding the levels of divergence between ara and arb. Do they share functional domains ?

Screening of ara and arb knockout mutants (KOs)
By screening the medaka TILLING library, we identified founders possessing nonsense mutations in exon 6 of ara (S507X) and exon 4 of arb (L503X) (Supplementary Fig. 2a-c). These nonsense mutations lead to expression of the truncated protein products lacking the LBD that contains a key amino acid-substitution responsible for distinct transactivation responses of Ara and Arb in vitro 30 ( Supplementary Fig. 2). Supplementary Fig.1

KOs")
Comparative genome sequence analysis combined with the available ATAC-seq data in Medaka 52 shows that putative cis-regulatory sequences of ara and arb, respectively, were partially conserved in teleost species, but not between ara and arb, supporting their subfunctionalization and/or neofunctionalization ( Supplementary Fig. 11).

Main text, page 11, line 356-363 (Result section of "Differences in expression and intracellular localisation of the two Ars")
The Ara-KI adult males showed weak green fluorescence throughout the trunk and fins, and strong fluorescence in the regions adjacent to the pectoral, dorsal and anal fins. In contrast, the Arb-KI adult males showed a more restricted pattern of green fluorescence that localized primarily to the pectoral, dorsal and anal fins. (Fig. 6b). In the papillary processes of the anal fin, stronger green fluorescence was observed in Arb-KI than in Ara-KI males (Fig. 6b). These differences in the expression patterns of Ara and Arb appear to be consistent with the results obtained from the comparative analysis of their cis-regulatory sequences.

Supplementary Fig. 11
Comparison of cis-regulatory sequences of ar genes in teleost a, A 20-kb medaka genomic sequence corresponding to ara gene and the 5' upstream and 3'  chr10: 22,174,238−22,194,238), and non-coding ATAC-seq peak sequences associated with the medaka arb gene (Li, Y. et al., 2020 2 ; http://tulab.genetics.ac.cn/medaka_omics/) using MultiPipMaker. Note that the ATAC-seq peaks associated with the medaka arb gene localize only within this 20-kb region. A blue arrow indicates arb with its transcriptional orientation. Green and yellow shadings indicate arb exons and introns, respectively. Only one peak region of ATAC-seq is conserved in the arb genome sequences of medaka, stickleback, and fugu (shaded in magenta) but not in the zebrafish ar or medaka ara genome sequences. These alignments suggest that cis-regulatory mechanisms of ara and arb, respectively, are partially conserved in the teleost species that retain both ar ohnologs, but not in the species that lack one or the other, and are not conserved between ara and arb. The genome sequences used here were downloaded from the UCSC Genome Browser and Ensembl Genome Browser 4, 5 .
We added the following paper in the References for Supplementary Information Supplementary Information, page 19 The authors included the discussion of identification of the downstream effector genes of Ars (Line420-line434), but I didn't find the relevant results for this investigation in the result section "Brain transcriptomic change in ar KOs". Maybe consider restructuring the presentation of these results and discussion.

Response: We appreciate the reviewer's comment.
We added the following sentences in the Result section of "Brain transcriptomic changes in ar KOs" .
Main text, page 10, line 329-335, Among these genes, expression level of neuropeptide B a (npba), which is known to regulate the female reproductive behaviour 49 , was 3.98 and 5.17 times higher in the arb KO and ar DKO males compared to WT males, respectively (Fig. 5g, i).
Additionally, expression level of hsd17b12a, whose product catalyses the transformation of estrone (E1) into E2 50 and 11-ketoandrostenedione (11KA4) to 11KT 51 , was significantly higher in the males of all ar KO strains compared to WT males (Fig. 5g-i).
Main text, page 10, line 338, We changed "oestron (E1)" to "E1", because the full name of abbreviation appeared in added sentences in line 333.
Main text, page 10, line 340-345, These results indicate that arb predominantly represses the npba expression, whereas both ar ohnologs are required for repressing hsd17b12a expression in males. This is likely the reason why 11KT level increased in ar DKO males. Such distinct roles for ar ohnologs in the regulation of Ar-biased gene expression in the brain may reflect the subfunctionalisation and/or neofunctionalisation of these ohnologs after the genome duplication event.
We added the following paper in References. More on this section, line295-lin296 , the authors wrote ' suggesting the occurrence of a small number of genes exhibiting low expression but having large effect on neuronal signal'. I find the support to this strong conclusion rather weakly supported, and it is not clear if it is based on the GO enrichment analysis or the fact that there are 290 DE genes? Is 290 genes fewer than expected compared to other studies analysing gene expression change related to behaviour? Could it be that the brain gene expression would be more or less exaggerated based on the context? Maybe consider rephrasing?
Response: We appreciate the reviewer's comment. We agree with the reviewer's suggestion. We could not detect the particular biological processes by Gene Ontology (GO) enrichment analysis, although we identified 290 genes that were differentially expressed between the WT and ar DKO males, indicating that whole brain transcriptome analysis provides insufficient resolution to identify the genes that regulates the courtship behaviour. Higher resolution analysis at the cellular level, an identification of the neurons that regulates the male reproductive behaviour and gene expression analysis in such specific neurons would be necessary to identify the genes that regulate male courtship display. Therefore, we have rephrased the sentences to explain the limitation of this study as follows.
Main text, page 9, line 318 -page 10, line 320 (Result section "Brain transcriptomic changes in ar KOs") Identification of genes regulating the male courtship display would require identification of neurons that control the male reproductive behaviour and high-resolution gene expression analysis in such neurons.
Line190 and Line 191 present contradictory ideas, potential a typo?
Response: We appreciate the reviewer's comment. We have amended the sentence of the corresponding part as follows.
Main text, page 6, line 208-209, These results indicate that all the mutant strains produce functional sperms that were capable of fertilising the eggs.
Line 354-line 358: I'm not quite following how ar signalling resolves sexual conflict in medaka, could the author elaborate on this idea? 'Acquisition of exaggerated male-specfic trains in the lineages leading to medaka' would be more convincing if the author comment on the level of sexual dimorphism in the lineages leading to medaka first.

Response: We greatly appreciate the reviewer's comment.
Because Ar signaling is dispensable for female fecundity but is indispensable for male fecundity by regulating the proper male specific reproductive behaviour and external morphology, we tried to suggest that androgen-dependent regulation would be able to solve the sexual conflict by regulating the male-biased phenotypes.

However recent analysis indicates that sex linkage which allows males and females
to carry different alleles on sex chromosomes is more effective than androgen regulation in the production of large sex differences in gene expression, and androgen-dependent regulation can contribute to temporary resolution of sexual conflict in stickleback 14 , Therefore, we cannot simply suggest that the androgen/Ar system resolves sexual conflict in medaka based on ar KO phenotypes. Therefore, we deleted "suggesting that the androgen/Ar system resolves sexual conflict in this species." from this sentence. Line 450-452: "resolved the pleiotropic function derived from their ancestral gene". I couldn't find relevant information in the manuscript for the function of ar in species that branched prior to TSGD to support this statement on 'ancestral' state. In our manuscript, we added the explanation about the protein property of Japanese eel Ars in the introduction and the phylogenetic tree of Ars in Supplementary Fig 1.

Main text, page 4, line 111-115 (Introduction)
Molecular evolutionary analysis of the teleost lineage has revealed the asymmetric evolution of ar ohnologs, including the accumulation of more novel substitutions in ara than in arb after the divergence of Elopomorpha, and that the lineage-specific loss of ara occurred independently in Otocephala such as zebrafish and Salmoniformes such as rainbow trout ( Supplementary Fig. 1) 24,25,[29][30][31] .

Main text, page 4, line 119-121 (Introduction)
We also found two key nonsynonymous base substitutions in the Ar hinge region and LBD, which are highly conserved among spiny-rayed fish (Acanthomorpha) Aras, including medaka and cichlid Aras but not in Japanese eel Ara 30 .
We also additionally wrote following sentences in the Discussion. Reviewer #3 (Remarks to the Author): Overall feedback: This manuscript describes a very thorough study in which the authors sought to identify the molecular properties of androgen receptor ohnologs underpinning the diversification of sexual characteristics in a fish, the Japanese medaka (Oryzias latipes). As such, this paper provides important insights into the mechanisms of radiation of teleost fishes driven by sexual selection.
I enjoyed reading this article. It covers an important topic using a study species that is less conventional than, for example, rodents, which have been used extensively in studies of sexual dimorphism. I also appreciate that the authors tested for effects of deficiency of androgen receptors alpha and beta on a broad range of morphological, reproductive, and behavioural traits (e.g. fin size and shape, tooth enlargement, spermatogenesis, courtship behaviour).
Because of the high standard and thoroughness of the study and manuscript, I have quite few comments and recommend a minor revision. However, I do think that these points will help to improve the manuscript.
Line-by-line feedback: Line 57: I am not sure about the use of the phrase 'behavioural attractiveness' here given that 'attractiveness' is perceived by the behavioural signal receiver, which depends on a whole host of other factors. Is there a more accurate term that could be substituted in here Response: We agree with the reviewer's comment. We have switched the phrase to "the reproductive behaviour eliciting female receptivity" in the revised manuscript.
We have amended the corresponding sentence as follows.

Main text, page 2, line 56-58 (Abstract)
ara was required for tooth enlargement and the reproductive behaviour eliciting female receptivity, while arb for male-specific fin morphogenesis and sexual motivation.

Main text, page 13, line 427-431 (Discussion)
Taken together, we revealed the differential roles of ar ohnologs associated with the unique sexual characteristics of teleosts-ara predominantly regulates the masculinisation of teeth and the reproductive behaviour eliciting female receptivity while arb plays essential roles in male-specific fin morphogenesis and sexual motivation.
Line 62: No capital is needed for 'first'. circular pattern in front of the female. If the female accepts the male, the male grasps her with his fins (termed "wrapping"), and they quiver together until eggs and sperm are released ("spawning").
If the male is not accepted, she either rapidly moves away from the male or assumes a rejection posture 38, 41 .
Line 269-272: The term 'wrapping' has not yet been defined and so the reader will not have proper context for this statement. It would be useful to earlier define this term and, in addition, briefly describe the typical mating behaviour of male and female medaka.
Response: We are grateful for this comment. The behaviour that the male grasps the female with his fins is termed "wrapping". Following this comment, we described the typical mating behaviours of both sexes in the Introduction as we have shown above.
Lines 682-683: 'The number of males escaping wrapping by the male was counted as the number of wrapping rejected by the female.' Again, these behaviours are not sufficiently described and so will be potentially confusing to readers. Wrapping behaviours that did not lead egg spawning were counted as wrapping rejections. If the wrapping led the spawning successfully, the period from the initiation of wrapping to the end of spawning was measured as a wrapping duration.
Main text, page 33, line 1136 (legend for Figure 5) "Duration of wrapping and spawning" has amended to "Duration of wrapping followed by spawning" Line 684-685: How were the behaviours recorded? Using a behaviour-logging software? If so, the name and version of the software should be provided. Also, was the observer blind to treatment while scoring trials?
Response: We appreciate the reviewer's comment. We recorded the behaviour using a digital video camera HDR-PJ800 (Sony, Tokyo, Japan) and then counted each behaviour by watching the recorded movie files. To distinguish the video files, line name indicated by number was recoded in video files, but we did not provide the information of each medaka lines to observer. within 1 hour of a behavioural trial, we judged the trial to be a draw. Effects of each ar mutation on the mate behaviour were statistically analysed using the chi-squared (χ 2 ) test of independence.
To measure the female fecundity of the ar KO strains, a female of WT, ara KO, arb KO, or ar DKO strains was transferred into a test tank with a WT male, and they were separated by a partition on the day before the test, as described above. After removing the separator in the morning of the test day, we analysed whether the females spawned the eggs. If the female successfully spawned, the total number of eggs obtained and their fertilisation rates were quantified. This test was repeated for 10 consecutive days by exchanging the males daily.
Line 713: I recommend that all data and code, including for behavioural trial data, are made publicly available alongside the article. In particular, uploading all of the data that would be required to re-run your analysis is crucial to making your research transparent.
Response: We appreciate the reviewer's comment. We uploaded all plot data in a single Excel file (Source data_MCOMMS-22-27469).
We are also ready to upload the huge video data of the behavior tests (１００GB). We would be happy to do it immediately if the editorial office could kindly tell us an appropriate server for Nature Communications.
We also revised the behavioural analysis data of ar DKO males for a more reliable comparison with those of WT and ar single KO males, because the number of their behavioural tests was smaller than those of WT and single ar KO males in our previous manuscript (13 tests in ar DKO, 52 tests in WT, 38 tests in ara KO, 33 tests in arb KO). In the revised data, we analyzed 43 mating tests for ar DKO males (30 minutes each), and found that ar DKO males took courtship behaviour in 6 mating tests, although the frequency of this behaviour was very few, once in the 4 tests and twice in the 2 tests. These results indicate that ar DKO males mostly, but not completely, abolished courtship displays. Therefore, we amended the corresponding texts of the revised manuscript as follows.

Main text page 8, line 273-276 (Result section" Differential effects of the two Ars on mating behaviours")
The ar DKO males mostly abolished courtship displays during behavioural testing, whereas the single mutant males displayed courtships in over 90% of the tests (Fig. 5a), indicating that either of the two ars is sufficient for this mating behaviour.

Main text page 11, line 382-385 (Discussion)
We demonstrated that ar DKO males mostly abolished courtship displays and lacked external sexual characteristics, such as masculinisation of fin morphology and pigment cell patterns, resulting in infertility.

Main text page 20, line 702-703 (Materials and Methods section "RNA isolation")
ar DKO males that lack courtship were anaesthetised after 5 min of mating with a WT female.
We revised the ar DKO data in Fig. 5a (Percentage of mating tests with different females in which a male exhibited courtship display within a 30 min).
We showed the WT data for comparison with those of ara KO or arb KO from the same litter.
We added the number of the behavioural tests that we analysed in the Figure legends for Fig. 1a and Fig. 5a as follows. We also summarized the number of the mating tests for each analysis in supplementary table 2. Main text, page 31, line 1055-1060 (Legend for Fig. 1a "Frequency of mating tests in which a WT female laid eggs within 30 min after mating") We used WT males (n = 11, total 60 mating tests) and ara KO males (n = 14, total 76 mating tests) of the same litter, and WT males (n = 6, total 42 mating tests) and arb KO males (n = 6, total 41 mating tests) of the same litter, and ar DKO males (n = 10, total 43 mating tests), as mating partners for the WT females. Details of mating tests utilized for this and following behavioural analyses were shown in supplementary Table 2.
Main text, page 33, line 1128-1132 (Legend for Fig. 5a "Percentage of mating tests with different females in which a male exhibited courtship display within 30 min") We used WT males (n = 11, total 59 mating tests) and ara KO males (n = 14, total 76 mating tests) of the same litter, and WT males (n = 6, total 40 mating tests) and arb KO males (n = 6, total 40 mating tests) of the same litter, and ar DKO males (n = 10, total 43 mating tests) as mating partners for the WT females.